Endothelial cell injury is considered to be the primary event in atherogenesis. We investigated the effects of materials released from white thrombi and hypercholesterolemia on endothelial cells and the vascular wall of rabbit aorta in vivo.
Polyethylene tubing was inserted into the ascending aorta of rabbits via the common carotid artery and placed for one, 4, and 24 weeks to induce vessel wall injury and thrombotic events. Then the non-injuried segments from the descending thoracic and abdominal aortas was morphologically examined. 3H-Thymidine uptake into endothelial cells and smooth muscle cells are also examined.
These aortas of experimental rabbits showed endothelial damage and ensuing endothelial replication. Modified smooth muscle cells were noted in the subendothelial layer, and in the 24-week experimental group, cellular intimal thickening was also induced. ³H-Thymidine uptake into the intima and media significantly increased in the experimental group. Administration of ASA, TXA₂ synthetase inhibitor (CV4151), and heparin partly reduced the endothelial damage and smooth muscle cell proliferation. Hypercholesterolemia also could cause endothelial damage, and a combination of materials released from white thrombi and hypercholesterolemia showed an additive effect on the aortic wall in vivo.

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Migration from the media, and proliferation in the intima, of the arterial smooth muscle cells are key events in the formation of intimal thickening. Migration and proliferation factors are essential in these processes. Paracrine secretion of migration and growth factors has already been reported in platelets, macrophages, granulocytes, and endothelial cells. However, the paracrine system explains only initial smooth muscle responses to the exogenous stimuli to the arterial wall because these factors disappear rapidly. For the further stimulation of migration and proliferation other factors that play a role must be identified. We propose here autocrine systems for migration and proliferation of smooth muscle cells.
Cultured rabbit aortic smooth muscle cells secreted both migration and growth factors for smooth muscle cells, named SDMF and SDGF, respectively. SDMF was a potent chemotactic factor with a molecular size about 400 kilodalton, the activity of which was several times as high as that of PDGF. This factor was different from known migration factors including PDGF and fibronectin. Intimal smooth muscle cells secreted more SDMF than medial smooth muscle cells.
SDGF was secreted from medial smooth muscle cells only after at least the 4th passage. Intimal smooth muscle cells secreted SDGF during very early passages. Activated endothelial cells modified smooth muscle cells so as to secrete SDGF. SDGF with a molecular weight of about 10 kilodalton was different from known growth factors, physicochemically, immunologically, and biologically.
The above results show that smooth muscle cells secrete new migration and growth factors in autocrine manners. This autocrine system must play a central role in the development of the Intimal thickening.

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Metabolism of glycated low density lipoprotein (glcLDL) was studied in a human hepatoma cell line (Hep-G2 cells). In the presence of 40μg/ml glcLDL, neither cultured human fibroblasts nor J774 cells accumulated cholesterol ester from ¹⁴C-oleate. In HepG2 cells, ¹⁴C-oleate was significantly incorporated into cholesterol ester in the presence of glcLDL, but the rates were small compared with those with native LDL. Studies using ¹²⁵I-labeled lipoprotein showed similar results. However, ¹²⁵I-glc-LDL degradation was similarly replaced by both native LDL and glcLDL, suggesting that the metabolic pathway is non-specific. Incorporation of ¹⁴C-oleate into cholesterol ester in the presence of 40μg/ml LDL was inhibited in HepG2 cells by IgG-C7, an antibody against LDL-receptors (0.7±0.4 n mole/mg prot. vs. 1.2±0.3 n mole/mg prot.). However, in the presence of 40μg glcLDL, the inhibition was not observed (0.4±0.1 n mole/mg prot. vs. 0.3±0.1 n mole/mg prot.). These results suggest that glcLDL is degraded in HepG2 cells through a metabolic pathway other than the classical LDL pathway.

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We report the clinical and biochemical characteristics of familial hyperalphalipoproteinemia associated with complete deficiency of plasma cholesteryl ester transfer protein (CETP) activity. The study subjects are 5 probands whose serum high density lipoprotein (HDL)-cholesterol levels ranged from 157 to 281mg/dl. Serum total cholesterol and apolipoproteins A-I, A-II, C-III and E were also elevated, while apolipoprotein B was decreased. Cholesteryl ester had accumulated in the HDL₂ fraction, which was large and rich in apolipoprotein E. The CETP activity was totally deficient in the probands, which was considered to be the cause of hyperalphalipoproteinemia. Nondenaturing polyacrylamide gradient gel electrophoresis, high performance liquid chromatography with gel permeation column and analytical ultracentrifugation disclosed small polydisperse low density lipoproteins (LDL) along with the markedly large HDL. In a patient whose HDL-cholesterol was 281mg/dl, lipoproteins with size range between LDL and HDL₂ were also detected. Lipoprotein lipase activity was very high in 4 of these patients, while hepatic triglyceride lipase activity was within normal limits. Restriction fragment length polymorphism (RFLP) of patients' DNA was examined in Southern blotting analysis using CETP-cDNA as a probe. The hybridization patterns in the probands were not significantly different from those of normal controls suggesting that there may be no gross alterations in the CETP gene.

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Recently, we identified two HDL binding proteins (Mr=120 K-Da and 100 K-Da) in rat and human liver plasma membranes by ligand blotting using ¹²⁵I-HDL₃, and purified these binding proteins. The detection method needed 3 or 4 days for its autoradiography.
Here, we report a method to visualize HDL binding proteins by ligand blotting using HDL₃, anti-apo A-I and HRPO-conjugated 2nd Ab. By this method, we identified the same binding proteins as those detected by ligand blotting using 125^I-HDL₃.
The advantages of this method are that we can visualize HDL binding proteins within 2 days and can use diluted serum as a ligand instead of radiolabeled purified HDL₃.

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A high incidence of restenosis at the site of successful PTCA is a major problem with this procedure. Among the various factors, thromboxane A₂ (TXA₂) and PGI₂ may have important roles on the restenosis in this mechanism. DP-1904 (DP), an imidazole derivative, is a potent selective TXA₂ synthetase inhibitor with prolonged activity. It also increases PGI₂ formation. In this study, the effect of DP on post-PTCA restenosis was compared with a placebo control taken from a recent trial conducted under an equivalent protocol. Angina and OMI patients receiving first elective PICA were enrolled in the study. Oral dosing of DP 600mg/day started at least 3 days before PTCA and continued until re-CAG at 3 months after PICA At re-CAG, restenosis was evaluated by lesion and patient based on the patency achieved at PICA. 1) Very good: no restenosis or expansion, 2) Good: <50% loss, 3) Poor: 50%≤loss<100%, 4) Very poor: 100% loss or more progressed stenosis before PICA. 19 patients (29 lesions) were evaluated in the DP group, and 15 (26 lesions) in the placebo group. DP: placebo evaluation of the stenotic lesion was: Very good 9: 5, Good 15: 10, Poor 2: 8 and Very poor 3: 3, indicating DP-s efficacy (Wilcoxon P=0.10). Evaluation by patient restenosis was: 26.3% in the DP group, 66.7% in the placebo group, showing DP's statistically significant efficacy over placebo (Wilcoxon P<0.05). No side effects were observed in the DP group. Plasma TXB₂/6-keto-PGF_1α ratio showed preferable changes during DP dosing: pre-dosing (2.58±0.82pg/ml), pre-PICA (0.60±0.13), immediately post-PICA (0.57±0.09) and 3 months post-PICA (0.84±0.09).

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Recently we have reported that an impairment of very low density lipoprotein (VLDL)-triglyceride (TG) catabolism is responsible for elevated plasma TG concentration in puromycin-induced nephrotic rats and that the half life of VLDL-TG from nephrotic rats in plasma was significantly longer than that from normal rats reinjected in normal recipient rats. We therefore analyzed the composition of VLDL in nephrotic rats to determine what's compositional change of VLDL particles may be associated with the defect in VLDL-TG catabolism. Polyacrylamide gel electrophoresis revealed that the relative proportion of apoprotein (apo) B 100+95/48 was significantly increased and the relative ratio of apo E to apo C was significantly decreased on nephrotic VLDL particles. No significant changes of apo C subspecies between nephrotic and normal VLDL was observed by the analysis of isoelectric focusing gel. The ratio of TG or cholesterol to apoprotein in VLDL was significantly increased in nephrosis, suggesting a larger particle size than the normal. These results suggest that compositional abnormalities of apoproteins and lipids is partially associated with the delayed clearance of nephrotic VLDL in plasma circulation.

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The clinical and biochemical features of the heterozygotes of lipoprotein lipase deficiency have not been fully understood, because the diagnosis of the heterozygote state has not been established. Babirak et al. recently reported that combined measurements of postheparin plasma LPL activity and of LPL mass allowed identification of the heterozygote state for LPL deficiency, some of which were found to have mild lipoprotein abnormalities.
Here we described a case of the heterozygote who exhibited chylomicronemia and was diagnosed by measurements of LPL activity and mass.
The patient was a male infant, who had multiple anomalies and growth retardation. Chylomicronemia was observed at six months of age. Measurements of serum lipids and apolipoprotein showed extreme hypertriglyceridemia (maximally 8, 280mg/dl at eleven months of age) and high apo C-II level. These clinical course and laboratory data were compatible with LPL deficiency, but postheparin LPL activity measured with artificial substrate and LPL mass measured in an enzymelinked immunoassay (ETA) were 58% and 27% of their normal values, respectively. This data accorded with the heterozygote state of LPL deficiency.
Measurements of LPL activity and mass also revealed that his mother and two sisters were heterozygotes, who were grossly normolipidemic. Their LPL activity was lower than the proband, but their LPL mass was greater than his. So in this family, LPL mass seemed to correlate better with LPL activity in vivo.
In any case, his LPL activity and mass were too great to cause chylomicronemia. Thus, it may be reasonable to speculate that other factors had temporally inhibited his LPL activity, causing transient chylomicronemia. We were not able to identify these factors, but production of LPL inhibitor as a consequence of bacterial infection may partly explain why he exhibited chylomicronemia.
This case showed that heterozygotes of LPL deficiency present severe chylomicronemia under certain conditions. It also suggests that their lipoprotein abnormalies have yet to be determined.

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γ-Oryzanol, known as an antiatherosclerotic and an antihyperlipidemia agent, prevents lipid peroxides from forming and accumulating in the vascular wall.
In this experiment, the effects of γ-Oryzanol and ferulic acid (a metabolite of γ-Oryzanol) on prostaglandin metabolism in the vascular wall and platelet were studied.
1) The effect of ferulic acid on PGI₂ production by cultured aortic endothelial cells was evaluated in the presence of histamine (10^-5 M) or Ca ionophore A23187 (5×10^-6 M) which stimulates PGI₂ synthesis. Ferulic acid significantly enhanced the 6-keto-PGF_1α (a stable metabolite of PGI₂; 6KF) production stimulated by histamine or Ca ionophore A23187 in a dose dependent manner, and maximal enhancement was observed at a ferulic acid dose of 200ng/ml. The mean percent increase in 6KF with 200ng/ml of ferulic acid was almost 66% with either stimulant. However, ferulic acid alone did not affect 6KF production at any dose.
2) The effects of ferulic acid on platelet aggregation and platelet thromboxane B₂ (a stable metabolite of thromboxane A₂; TXB₂) production induced by thrombin (0.03U/ml) were also evaluated using washed platelets from healthy volunteers (n=4). Ferulic acid significantly inhibited the platelet aggregation and the platelet TXB₂ production in a dose dependent manner, and its inhibitory effect reached a plateau at a ferulic acid dose of 200ng/ml.
3) γ-Oryzanol was orally given to healthy volunteers (n=9) at a daily dose of 300mg for 2 weeks. Levels of 6KF and TXB₂ in the plasma and the ADP-induced platelet aggregation rate were measured after 1 and 2 weeks of administration. The ratio of 6KF: TXB₂, which mainly regulates the function of platelets in vivo, was significantly elevated by the 2-week administration, though the individual levels of 6KF and TXB₂ in the plasma were not changed significantly. In addition, the platelet aggregation induced by ADP (4μM) was significantly inhibited by the 2-week administration.
These results indicate that γ-Oryzanol and its metabolites can be effective agents for the prostaglandin metabolism in the vascular wall and platelets in vivo, preventing the development of vascular lesions such as atherosclerosis.

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The cholesterol and protein concentrations in apoprotein A-I and B-100 containing particles after oral glucose injection (0, 30, 60, 120min(s).) isolated using selected-affinity columns with monoclonal antibodies from 13 diabetic patients and 14 non-diabetic subjects were analyzed. Changes in total plasma cholesterol levels after oral glucose injestion in the 2 groups were not significant. However, cholesterol and protein contents in apoprotein B-100 particles after oral glucose injection were significantly decreased (p<0.05) in the 2 groups. Changes in cholesterol content in apoprotein A-I particles after oral glucose injection (30, 60min(s).) were significantly decreased (p<0.05) in the diabetic patients group, but were not significant in the non-diabetes group. The changes in protein content in apoprotein A-I particles after oral glucose injection in 2 groups were not significant. The data indicated that protein contents in apoprotein A-I or B-100 containing particles after oral glucose injection relatively increased and cholesterol concentrations in other lipoprotein fractions might increase after oral glucose injection especially in the diabetic patients.

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We experienced a case of familial hypercholesterolemia (FH) which received simultaneous coronary artery bypass grafting and replacement therapy of an abdominal aortic aneurysm (AAA).
In 1977 FH was diagnosed in a 66-year-old man with serum cholesterol of 344mg/dl, triglyceride of 101mg/dl, and achilles tendon thickness of 14mm. In 1983 coronary angiography detected 75% stenosis in segment 7 of the left anterior descending artery (LAD). In 1985 abdominal CT scan detected AAA with a diameter of 4cm, which had increased in size by 1988.
We performed re-examinations of abdominal CT scan, abdominal ultrasonography, and angiography. The results showed that the AAA had increased to a diameter of 7cm with a thrombus inside, and that the coronary artery atherosclerosis had progressed diffusely, with progression of the LAD proximal lesion resulting in an increase from 22 to 26 in the coronary sclerotic index. Accordingly, coronary artery bypass grafting to LAD segment 8 and replacement therapy of AAA were carried out simultaneously. In the case of FH, systemic atherosclerosis progresses so easily that it is imperative that close attention be paid not only to lesions in the coronary arteries but also atherosclerotic lesions elsewhere.

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